Determination of Acrylamide: Are You Avoiding Isobaric Interferences?

Importance of the Topic

Acrylamide is a processing contaminant formed during high-temperature cooking via the Maillard reaction. It poses significant toxicological risks, including neurotoxicity, genotoxicity, carcinogenicity, and reproductive toxicity. Regulatory bodies such as the European Commission have set benchmark levels and mitigation requirements to control acrylamide in foods. Reliable measurement of trace levels in diverse matrices like potato chips, coffee, and baby food is therefore essential for food safety and regulatory compliance.

Objectives and Study Overview

This application note revisits a previously published LC-MS/MS method for acrylamide to address potential isobaric interferences reported for N-acetyl-β-alanine, 3-aminopropanamide, and lactamide. The study aimed to:

• Confirm in-source fragmentation of the three compounds to the same precursor and product ions as acrylamide
• Evaluate clean-up protocols for removal of interferences
• Verify chromatographic resolution under existing UPLC conditions
• Validate method accuracy using FAPAS coffee reference material

Methodology and Instrumentation

A modified QuEChERS extraction was performed on processed food samples, followed by two clean-up approaches:

• Dispersive SPE (dSPE)
• Oasis MCX pass-through SPE

Chemical separation and detection used:

• UPLC column: ACQUITY UPLC HSS C18 SB
• Mobile phase A: water with 0.1% formic acid (pH-controlled), B: methanol
• Mass spectrometer: Xevo TQ-S micro Triple Quadrupole
• Acquisition mode: RADAR (simultaneous MRM and full-scan MS)

Main Results and Discussion

RADAR scans of N-acetyl-β-alanine, 3-aminopropanamide, and lactamide confirmed in-source fragmentation to m/z 72, which then produced product ions at m/z 55 and 27—the same transitions used for acrylamide quantification. Neither dSPE nor MCX SPE fully removed these interferences, making chromatographic separation essential. Under the published pH-controlled LC conditions, all three compounds were baseline resolved from the acrylamide peak. When formic acid was omitted (neutral pH), N-acetyl-β-alanine shifted retention time and 3-aminopropanamide was not retained, risking co-elution. Analysis of FAPAS coffee reference material (TYG010RM) yielded acrylamide concentrations of 248–250 µg/kg, in line with the certified 249 µg/kg value, demonstrating method accuracy and selectivity.

Benefits and Practical Applications

The validated approach offers:

• Rapid and cost-effective quantification of acrylamide in complex food matrices
• High selectivity through chromatographic resolution of isobaric interferences
• Robust performance across dispersive and pass-through SPE clean-up
• Compliance with EU regulatory benchmarks

Future Trends and Opportunities

Advancements may include:

• Integration of high-resolution mass spectrometry to further enhance selectivity
• Automated sample preparation workflows for higher throughput
• Miniaturized extraction techniques to reduce solvent consumption
• Application to a broader range of food matrices and emerging processing contaminants

Conclusion

The Waters acrylamide kit, combined with a pH-controlled UPLC method and dual SPE clean-up options, reliably separates acrylamide from isobaric interferences and delivers accurate quantification in food samples. Critical control of mobile phase pH ensures baseline resolution of potential co-eluting compounds, confirming method suitability for regulatory monitoring.

Reference

1. EFSA CONTAM Panel, 2015. Scientific Opinion on Acrylamide in Food. EFSA Journal 13(6):4104.
2. Commission Regulation (EU) 2017/2158 of 20 November 2017. Establishing Mitigation Measures and Benchmark Levels for the Reduction of Acrylamide in Food.
3. Waters Corporation, 2019. Determination of Acrylamide in Processed Foods using ACQUITY UPLC I-Class and Xevo TQ-S micro (p/n: 720006495).
4. Desmarchelier A., Hamel J., Delatour T., 2020. Source of Overestimation in the Analysis of Acrylamide in Coffee by Liquid Chromatography Mass Spectrometry. Journal of Chromatography A 1610:460566.